NAD(+) use is an ancestral trait of isocitrate dehydrogenase (IDH), and the NADP(+) phenotype arose through evolution as an ancient adaptation event. However, no NAD(+)-specific IDHs have been found among type II IDHs and monomeric IDHs. In this study, novel type II homodimeric NAD-IDHs from Ostreococcus lucimarinus CCE9901 IDH (OlIDH) and Micromonas sp. RCC299 (MiIDH), and novel monomeric NAD-IDHs from Campylobacter sp. FOBRC14 IDH (CaIDH) and Campylobacter curvus (CcIDH) were reported for the first time. The homodimeric OlIDH and monomeric CaIDH were determined by size exclusion chromatography and MALDI-TOF/TOF mass spectrometry. All the four IDHs were demonstrated to be NAD(+)-specific, since OlIDH, MiIDH, CaIDH and CcIDH displayed 99-fold, 224-fold, 61-fold and 37-fold preferences for NAD(+) over NADP(+), respectively. The putative coenzyme discriminating amino acids (Asp326/Met327 in OlIDH, Leu584/Asp595 in CaIDH) were evaluated, and the coenzyme specificities of the two mutants, OlIDH R(326)H(327) and CaIDH H(584)R(595), were completely reversed from NAD(+) to NADP(+). The detailed biochemical properties, including optimal reaction pH and temperature, thermostability, and metal ion effects, of OlIDH and CaIDH were further investigated. The evolutionary connections among OlIDH, CaIDH, and all the other forms of IDHs were described and discussed thoroughly.